Project Summary/Abstract Tendons are difficult to heal and tendinopathies are painful, leading to absenteeism at work and negatively affecting quality of life. The more we know about basic tendon cell (i.e., tenocyte) biology, the better we will be prepared to prevent and treat tendon disorders. Basic tenocyte knowledge is essential for applied tendon research (e.g., tendon engineering approaches to treat tendinopathies). In the body, calcium (Ca2+) is ubiquitous and crucial for several important aspects of cell life and its role is defined by intracellular spatiotemporal variations controlled by ion channels that move the Ca2+ between the cytosol and cellular stores/extracellular environment. One of these channels, the dihydropyridine receptor (DHPR), known as a voltage-dependent Ca2+ channel, would be mechano-dependent in non-excitable cells, that is, stimulated by cell ?stretching? (strain). Not much is known about DHPR function and its relationship to others aspects of calcium metabolism in tenocytes. Our objective is to investigate how DHPR function is affected in vitro by different conditions of tenocyte strain, not only in terms of magnitude but also frequency. Since the incidence of tendinopathies increase as we age, we will also look into how aging affects DHPR function. In the first Aim of this proposal we will determine the relationship between DHPR and two well-known ways that cells use to control their intracellular calcium concentration: Store-Operated Calcium Entry (SOCE) and calcium oscillations. Relevant questions are: are SOCE and calcium oscillations affected by DHPR function? Is this effect dependent on tenocyte strain conditions? How are the answers to those questions affected by aging? Oxidative stress is another essential part of the cell life, sometimes necessary for cellular homeostasis, sometimes the cause of cellular damage. Furthermore, the cells response to oxidative stress, which is particularly important in mechanosensitive cells, is interconnected with their calcium metabolism. This way, in the second Aim we will study the role of DHPR in the response of tenocytes to oxidative stress. For the same reasons of Aim 1, the relationship between DHPR and oxidative stress in tenocytes will be explored under different conditions of cell strain and age. In order to assess DHPR function in tenocytes, we will perform the proposed assays after an initial period of incubation with well- known inhibitor or agonist (for control, no inhibition or stimulation of the channel). We will also inhibit DHPR at the gene expression level with siRNA. After this work, when we contrast and compare the different results, we expect to reach a better knowledge of the role of DHPR in tenocytes, and consequently, in tendon homoeostasis. This proposal has the potential to cause a paradigm shift in the way we consider calcium intracellular signaling in tenocytes, how we see the importance of DHPR for tenocyte homeostasis, and how DHPR could influence some of the studies on tenocytes performed in usual monolayer culture (i.e., tenocytes under no strain). It could also unveil a new path for future therapeutic interventions to treat tendinopathies.